1. Field of the Invention
The present invention relates to a method for efficiently producing a transgenic plant using genetic engineering techniques, and a vector used for the method.
2. Brief Description of the Background Art
When a transgenic plant is produced by introducing a desired gene into a plant of interest, the following 3 steps are always required: (1) introduction of the desired gene into a plant cell, (2) selection of a plant tissue (desired gene-introduced tissue) containing desired gene-introduced cells, and (3) redifferentiation of a plant from the selected plant tissue. In the selection of a desired gene-introduced tissue among these steps, generally, it is not easy to select such tissue using expression of the desired gene alone as an index at the stage of cell culturing, so that the desired gene is introduced into a plant cell together with a selectable marker gene whose expression can be easily detected, and the desired gene-introduced tissue is selected based on the presence or absence of the expression of the selectable marker gene. The selectable marker gene used practically and frequently includes genes relating to drug resistances such as a kanamycin-resistant gene (NPTII: neomycin phosphotransferase gene) and a hygromycin-resistant gene (HPT: hygromycin phosphotransferase gene) which provide resistance to antibiotics, a sulfonylurea-resistant gene (ALS: acetolactate synthase gene) which provides resistance to agricultural chemicals, and the like.
When a gene relating to a drug resistance is used as the selectable marker gene, cells after gene introduction treatment are cultured using a medium containing such a drug, the presence or absence of the selectable marker gene, namely resistance to the drug, is evaluated, and selection is carried out using the evaluation as an index. Since such a drug originally has toxicity to plant cells, plant cells into which the selectable marker gene (so the desired gene) is not introduced die when cultured using such a medium. However, even if the resistance is present in this case, namely even if a plant cell can grow in the presence of such a drug, it is a matter of degree, so that bad influences of the culturing in the presence of such a drug upon plant cells cannot be avoided, thus actually causing problems such as reduction of a growth ratio and a redifferentiation ratio of desired gene-introduced tissue accompanied by the activity reduction of the plant cells.
In order to solve the problems, a novel vector for gene introduction into a plant and a gene introduction method into a plant using the vector have been proposed in JP-A-9-154580. When a gene is introduced using the vector and the method, desired gene-introduced tissue can be selected using only a morphological change in plant tissue after the gene introduction as an index, without using a drug which reduces the activity of plant cells.
In the above vector and method disclosed in JP-A-9-154580, a morphological abnormality induction gene such as a plant hormone synthetic gene is used as the selectable marker gene, and the desired gene-introduced tissue can be obtained as an adventitious bud or an adventitious root from a tissue after the gene introduction. However, among the adventitious buds and adventitious roots obtained in this manner, those to which the desired gene was not introduced (hereinafter also referred to as “escape”) were present in a fairly large amount. It seems that a plant hormone and the like produced in a cell to which an morphological abnormality induction gene has been introduced together with the desired gene are transferred into its peripheral cells and have influence thereon, and tissue showing morphological abnormality is differentiated and proliferated also from the gene-unintroduced cells which has been influenced. It is generally considered to be difficult to introduce a gene into plants, and the adventitious bud originally has a poor redifferentiation ratio in the plants. Furthermore, selection efficiency of desired gene-introduced tissue is very poor due to the presence of this escape, so that improvements of the redifferentiation ratio and the selection efficiency have been particularly desired.